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Dusseldorf, Germany

Festel G.,Autodisplay Biotech GmbH | Maas R.,Autodisplay Biotech GmbH
Journal of Commercial Biotechnology

An increasing number of chemicals and materials like base chemicals and polymers as well as high value products such as consumer chemicals and specialty chemicals are produced using biotechnology in one or more of the process steps. In 2010 the sale volume of biotechnology products was around 92 billion Euro worldwide. Sales are estimated to increase to around 228 billion Euro in 2015 and to around 515 billion Euro in 2020. On a sector level the largest market potential lies in the production of biopolymers and active pharmaceutical ingredients. Aas a rule commercial development is mainly driven by multinational enterprises whereas small and medium enterprises contribute primarily to the technological development. Especially the latter group faces several challenges during their development. These mainly concern business models and growth strategies as well as financing strategies and resources. Investors have not yet fully identified the area of industrial biotechnology as an attractive investment field but they could become a major capital source as they start to understand more the potential of industrial biotechnology. Source

Kranen E.,Autodisplay Biotech GmbH | Detzel C.,Autodisplay Biotech GmbH | Weber T.,Henkel AG | Jose J.,University of Munster
Microbial Cell Factories

Background: Lipases including the lipase from Burkholderia cepacia are in a main focus in biotechnology research since many years because of their manifold possibilities for application in industrial processes. The application of Burkholderia cepacia lipase for these processes appears complicated because of the need for support by a chaperone, the lipase specific foldase. Purification and reconstitution protocols therefore interfere with an economic implementation of such enzymes in industry. Autodisplay is a convenient method to express a variety of passenger proteins on the surface of E. coli. This method makes subsequent purification steps to obtain the protein of interest unnecessary. If enzymes are used as passengers, the corresponding cells can simply be applied as whole cell biocatalysts. Furthermore, enzymes surface displayed in this manner often acquire stabilization by anchoring within the outer membrane of E. coli.Results: The lipase and its chaperone foldase from B. cepacia were co-expressed on the surface of E. coli via autodisplay. The whole cell biocatalyst obtained thereby exhibited an enzymatic activity of 2.73 mU mL-1 towards the substrate p-nitrophenyl palmitate when applied in an OD578 =1. Outer membrane fractions prepared from the same culture volume showed a lipase activity of 4.01 mU mL-1. The lipase-whole cell biocatalyst as well as outer membrane preparations thereof were used in a standardized laundry test, usually adopted to determine the power of washing agents. In this test, the lipase whole cell biocatalyst and the membrane preparation derived thereof exhibited the same lipolytic activity as the purified lipase from B. cepacia and a lipase preparation which is already applied in commercial washing agents.Conclusions: Co-expression of both the lipase and its chaperone foldase on the surface of E. coli yields a lipid degrading whole cell biocatalyst. Therefore the chaperone supported folding process, absolutely required for the lipolytic activity appears not to be hindered by surface display. Furthermore, the cells and the membrane preparations appeared to be stable enough to endure a European standard laundry test and show efficient fat removal properties herein. © 2014 Kranen et al.; licensee BioMed Central Ltd. Source

Detzel C.,Heinrich Heine University Dusseldorf | Maas R.,Heinrich Heine University Dusseldorf | Maas R.,Autodisplay Biotech GmbH | Jose J.,Heinrich Heine University Dusseldorf

In the present study, a whole cell biocatalyst for the synthesis of (R)-mandelic acid from mandelonitrile was constructed. For this purpose, nitrilase from Alcaligenes faecalis subsp. faecalis ATCC 8750 was displayed on the surface of Escherichia coli by using Autodisplay. Localization of the nitrilase in the cell envelope of E. coli was monitored by SDS-PAGE and surface exposure was verified by its accessibility to externally added protease. The whole cell biocatalyst converted up to 2.6mM of (R)-mandelic acid under optimum conditions at pH7.5 and 45°C within 24h (1mL culture, OD 578=10). By using chiral HPLC, the ee value of the product was determined to be >99%. The surface displayed nitrilase showed an apparent K m value of 3.6mM and an apparent V max value of 1nmolmin -1mL -1 when a bacterial suspension of OD 578 3 was used. Substrate inhibition by benzaldehyde was similar to that of the free enzyme. The whole-cell biocatalyst retained 55% of its initial (R)-mandelic acid production after 5 cycles of repeated use, and could be stored at -70°C for 180d without a substantial loss of activity. In addition the whole cell biocatalyst converted 9.3mM phenylacetonitrile within 16h. © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim. Source

Kranen E.,Heinrich Heine University Dusseldorf | Steffan N.,University of Marburg | Maas R.,Autodisplay Biotech GmbH | Li S.-M.,University of Marburg | Jose J.,Heinrich Heine University Dusseldorf

The following study depicts the development of a whole cell biocatalyst for the prenylation of indole derivatives. For this purpose the prenyltransferase FgaPT2 from Aspergillus fumigatus was displayed on the surface of Escherichia coli cells by using Autodisplay. The presence of the prenyltransferase in the outer membrane was detected by using SDS-PAGE and Western Blot after the proteins of the outer membrane were isolated. The orientation of the prenyltransferase towards the outside of the cells was investigated by accessibility testing with externally added proteases. The FgaPT2 whole cell biocatalyst converted up to 250μM of indole-3-propionic acid, approximately 25% of the substrate used in the assay (100μL sample, OD 578=40). Another indole substrate, L-β-homotryptophan was also investigated and a conversion of 13% was determined. By optimizing the assay conditions the conversion rate could be raised to approximately 30% of indole-3-propionic acid during a 24h incubation time at 20°C. The whole cell biocatalyst endured a storage period of one month at 8°C without any detectable loss in activity. Reusability was confirmed by recycling the biocatalyst. After three cycles of consecutive use, the whole cell biocatalyst retained a conversion rate of 46% of indole-3-propionic acid and 23% of L-β-homotryptophan after the third cycle. © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim. Source

The present invention relates to a method for the surface display of a recombinant polypeptide on the surface of a host cell, said method comprising the steps: (a) providing a host cell transformed with a nucleic acid fusion operatively linked with an expression control sequence, said nucleic acid fusion comprising: (i) a portion encoding a signal peptide, (ii) a portion encoding the recombinant polypeptide to be displayed, (iii) a portion encoding a transmembrane linker, and (iv) a portion encoding the trans porter domain of an EhaA protein, and (b) culturing the host cell under conditions wherein the nucleic acid fusion is expressed and the expression product comprising the recombinant polypeptide is displayed on the surface of the host cell.

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